Identification of immunogenic HLA class I and II neoantigens using surrogate immunopeptidomes

Neoantigens arising from somatic mutations are tumor specific and induce antitumor host T cell responses. However, their sequences are individual specific and need to be identified for each patient for therapeutic applications. Here, we present a proteogenomic approach for neoantigen identification, named Neoantigen Selection using a Surrogate Immunopeptidome (NESSIE). This approach uses an autologous wild-type immunopeptidome as a surrogate for the tumor immunopeptidome and allows human leukocyte antigen (HLA)–agnostic identification of both HLA class I (HLA-I) and HLA class II (HLA-II) neoantigens. We demonstrate the direct identification of highly immunogenic HLA-I and HLA-II neoantigens using NESSIE in patients with colorectal cancer and endometrial cancer. Fresh or frozen tumor samples are not required for analysis, making it applicable to many patients in clinical settings. We also demonstrate tumor prevention by vaccination with selected neoantigens in a preclinical mouse model. This approach may benefit personalized T cell–mediated immunotherapies.

The PDF file includes:

Figs. S1 to S7 Legends for tables S1 to S5
Other Supplementary Material for this manuscript includes the following: Tables S1 to S5 3.1%   A, Blueprint of a tandem IVTT vector.One vector contained up to 10 neoantigen units (shown as A to J), each unit consisting of a neoantigen coding sequence (shown as NeoAg) with an upstream T7 promoter, ribosome binding site (RBS), start codon and FLAG sequence, and a downstream stop codon and 3'UTR stem loop.B, Preparation of IVTT products.For the 1st round screening, a mixed pool of neoantigen peptides was generated from each vector by IVTT.For the 2nd round screening, each neoantigen sequence was digested with the indicated restriction enzymes, separated and extracted by electrophoresis, and separately transcribed and translated by IVTT.C, Flow cytometry of CD8 + bulk CRC135 TIL products for the 1st round of screening.Intracellular IFNγ production and CD107a surface expression were measured in response to T2 cells pulsed with the IVTT products of the tandem IVTT vectors (V01 to V13).D, Flow cytometry of CD8 + bulk CRC135 TIL products for the 2nd round of screening.Intracellular IFNγ production and CD107a surface expression were measured in response to T2 cells pulsed with the IVTT products of vector V02 consisting of 10 unique neoantigens (N011 to N020).
Numbers indicate frequency (%) of IFNγ + CD107a + cells in CD3 + CD8 + cells.A, Flow cytometry of CD8 + spleen cells after the administration of KSL9 vaccine alone (n = 7), PD-1 antibody alone (n = 9), or both (n = 10) with a control group (n = 4).Intracellular IFNγ production and CD107a surface expression in response to KSL9 peptides were measured as described in the Methods section.B, Bar graph representing the frequency of KSL9reactive CD8 + T cells shown in Fig. S7A.
Fig. S1 | Neoantigen (KRV9)-reactive CD8 + T cell clones.A, Gating strategies to assess the neoantigen reactivity of CRC135 bulk TILs using flow cytometry.B, Flow cytometry of CRC135 bulk TILs.Numbers in the rectangles indicate the proportion of 4-1BB + cells in CD3 + CD8 + cells.TILs were analyzed in the presence of T2 cells pulsed with the indicated peptides.Data are representative of two independent experiments.C, IFNγ ELISPOT assay of KRV9-reactive CD8 + T cell clones (4B6, 5B9, 2D9, 6F2, 2D6, 2E7, and 4H11) with the same TCRαβ clonotype (TCR1) sorted from CRC135 bulk TILs in response to T2 cells pulsed with 100 nM of the indicated peptides.Data are representative of four independent experiments.D, Frequency of IFNγ + CD107a + cells recognizing the indicating peptide in the 4H11 clone.Data are representative of two independent experiments.E, Distribution (bar chart) and frequency (pie chart) of TCRα clonotypes in CRC135 bulk TILs (136 clonotypes in total).TCRα clonotype of the KRV9-reactive CD8 + T-cell clones (TCR1) is shown in red.
Fig. S3 | Neoantigen (KVI10)-reactive CD8 + T cell clones.A, Gating strategies to assess the neoantigen reactivity of UTE003 bulk TILs by flow cytometry.B, IFNγ ELISPOT assay of KVI10-reactive CD8 + T cell clones with different TCRαβ clonotypes derived from UTE003 TILs in the presence of 1 μM of the indicated peptides.Data are representative of two independent experiments.C, IFNγ ELISPOT assay of clones 47 and 24 against a panel of LCLs pulsed with 1 μM KVI10 peptide, indicating the HLA-A*02:01 restriction of T cell responses to KVI10.Data are representative of two independent experiments.